Published May 1, 1979
Amplitude, Kinetics, and Reversibility
of a Light-Induced Decrease in
Guanosine 3',5'-Cyclic Monophosphate
in Frog Photoreceptor Membranes
MICHAEL
L. WOODRUFF
and M. D E R I C
BOWNDS
From the Laboratory of Molecular Biology and the Department of Zoology, University of
Wisconsin, Madison, Wisconsin 53706
INTRODUCTION
In vertebrate rod photoreceptors biochemical machinery for excitation and
a d a p t a t i o n is c o n t a i n e d w i t h i n t h e o u t e r s e g m e n t , a c y l i n d r i c a l o r g a n e l l e w h i c h
J. GEN. PHYSIOL.~) The Rockefeller University Press - 00"22-1295/79/05/062912551.00
Volume 73 May 1979 629-653
629
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A B S T R A C T T h e concentration of guanosine 3',5'-cyclic m o n o p h o s p h a t e (cyclic
GMP) has been e x a m i n e d in suspensions of freshly isolated frog rod outer
segments using conditions which previously have been shown to maintain the
ability o f outer segments to p e r f o r m a light-induced permeability change (presence
o f calf serum, anti-oxidant, and low calcium concentration). Illumination causes a
r a p i d decrease in cyclic GMP levels which has a half-time - 1 2 5 ms. With light
exposures that bleach less than 100 rhodopsin molecules in each rod outer segment,
at least 104-105 molecules of cyclic GMP are hydrolyzed for each rhodopsin
molecule bleached. H a l f o f the total cyclic GMP in each outer segment, ~2 • 107
molecules, is contained in the light-sensitive pool. If outer segments are exposed to
continuous illumination, using intensities which bleach between 5.0 x 101 and 5.0
x 104 r h o d o p s i n molecules/outer segment per second, cyclic GMP levels fall to a
value characteristic for the intensity used. This suggests that a balance between
synthesis and d e g r a d a t i o n of cyclic GMP is established. This constant level appears
to be regulated by the rate of bleaching rhodopsin molecules (by the intensity o f
illumination), not the absolute n u m b e r o f r h o d o p s i n molecules bleached. After
brief exposure to light of varying intensities, cyclic GMP concentration returns to
near the value observed in unilluminated outer segments within 30-60 s. T h e
recovery is most r a p i d after dim illumination. Decreases in cyclic GMP levels
induced by light s u p e r i m p o s e d on b a c k g r o u n d illumination are r e d u c e d by an
a m o u n t proportional to the intensity of the b a c k g r o u n d light. Background light
a p p e a r s to have no effect on the sensitivity of the light-dependent cyclic GMP
decrease. Calcium ions lower the level of cyclic GMP without influencing either the
final level to which cyclic GMP is r e d u c e d by illumination or the light sensitivity o f
the reduction. These data, together with those of a previous p a p e r presenting
several correlations between cyclic GMP levels and light suppressible ionic permeability o f isolated frog rod o u t e r segments ( W o o d r u f f et al., 1977. J. Gen. Physiol.
69: 667-679), suggest that cyclic GMP is involved in visual transduction, perhaps
mediating between p h o t o n absorption and the permeability decrease.
Published May 1, 1979
630
T H E J O U R N A L O F GENEl~dLL P H Y S I O L O G Y 9 V O L U M E 7 3 "
1979
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in the amphibian is 6-8/~m in diameter and 50-80/~m in length. The rod outer
segment is a modified cilium composed of a plasma membrane which encloses a
stacked series of 1,500-2,000 disc membranes. When light strikes one of the - 3
x 10~ rhodopsin molecules in the disc membranes, a transient suppression of
the permeability of the plasma membrane occurs. This permeability decrease
causes a hyperpolarization of the plasma membrane. The disc membranes are
physically separate from the plasma membrane, and for this reason as well as
others (see Hagins, 1972; Tomita, 1970), it is commonly assumed that an internal
transmitter substance must transfer information from the site of photon
absorption in the disc membranes to the permeability mechanism of the plasma
membrane. The concentration of the presumed transmitter might increase or
decrease in response to illumination, and be regulated by trans-disc membrane
fluxes or by enzymic synthesis and breakdown. Ions, small organic molecules,
and proteins are all potential transmitters. Considerable interest and effort has
focused on the specific suggestion that light absorption by rhodopsin causes the
release of calcium ions stored within the disc membrane. This calcium is
assumed to bind to and inhibit the conductance mechanism of the plasma
membrane. Definitive evidence proving or disproving this hypothesis has not
been obtained (see Hagins and Yoshikami, 1977; Szuts and Cone, 1977).
In the past few years evidence has been accumulating that cyclic GMP may be
an important molecule in photoreceptor physiology. Illumination causes a
decrease in the level of cyclic GMP in photoreceptor cells (Orr et al., 1976;
Cohen et al., 1978) and in isolated outer segments (Fletcher and Chader, 1976;
Woodruff et al., 1977), apparently by activating the degradative enzyme, cyclic
GMP-phosphodiesterase (Miki et al., 1973, 1975; Keirns et al., 1975; Krishna et
al., 1976; Goridis and Weller, 1976; and Wheeler and Bitensky, 1977). The level
to which cyclic GMP is reduced reflects the amount of incident radiation
(Woodruff et al., 1977). Drugs which inhibit phosphodiesterase and increase
endogenous cyclic GMP levels also increase the ionic permeability of isolated
outer segments assayed in vitro (Brodie and Bownds, 1976), and cause depolarization of toad rods (Lipton et al., 1977 b). Woodruff et al. (1977) have recently
shown that cyclic GMP levels and the ionic permeability of frog rod outer
segments are suppressed over the same range of light intensity, and that
pharmacological agents affect both similarly. The cyclic GMP decrease triggered
by illumination was found to occur within 1 s, with several thousand cyclic GMP
molecules disappearing for each rhodopsin molecule bleached.
This paper reports further studies of the rapid cyclic GMP decrease in
isolated outer segments using conditions which maintain their transduction
mechanism (Bownds and Brodie, 1975; Brodie and Bownds, 1976). The questions asked here are: (a) how rapidly is cyclic GMP decreased upon illumination?
(b) is the decrease reversed when illumination stops? (r Is the amplitude of the
cyclic GMP decrease regulated by the total number of rhodopsin molecules
bleached or the rate of their bleaching (by total illumination or light intensity)?
(d) What is the sensitivity of the decrease (i.e., how many cyclic GMP molecules
are hydrolyzed for each rhodopsin molecule bleached)? (e) Is the cyclic GMP
response desensitized ("light-adapted") by a prior exposure to illumination? and
(f) Do calcium ions influence the cyclic GMP decrease?
Published May 1, 1979
WOODRUff ANDBOWNDS Light-lnduced Decrease in Cyclic GMP: Characteristics
631
METHODS
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Bullfrogs (Rana catesbeiana), 10-15 cm in length, were used in these experiments. T h e
frogs were kept in light-tight holding tanks for 2-6 wk before use. Each animal was
force-fed - 5 g o f Purina Dog Chow (Ralston Purina Co., St. Louis, Mo.), vitamin
s u p p l e m e n t e d , three times a week. During this holding period the frogs received a 12-h
light-dark cycle, with intermittent illumination d u r i n g the light part of the photoperiod:
5 min o f d i m white light, 5 rain o f light flashes at a frequency o f one p e r second, and 5
min o f darkness. This regimen o f illumination was used to provide more retinal
stimulation (alteration o f light and d a r k adaptation) than would be provided by
continuous illumination. Frogs were sacrificed for experiments 1-6 h before the end o f
the 12-h d a r k period. All experimental manipulations were carried out in infrared
illumination, a h e a d - m o u n t e d IR converter (FJW. Industries, Mount Prospect, Ill.) being
used for visualization. Retinas were removed as described previously ( W o o d r u f f et al.,
1977) a n d gently rinsed in a modified frog Ringer's solution (115 mM sodium chloride,
2.5 mM potassium chloride, 10 mM Hepes buffer [N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid], 10% vol/vol undialyzed calf serum, 1 mM dithiothreitol and 3 mM
E G T A [ethylene glycoi-bis-(fl-aminoethyl ether) N,N'-tetraacetic acid], p H 7.5). This
modified Ringer's solution was used because it has been f o u n d to be optimal for
maintaining r o d o u t e r segment structure and function in vitro (Bownds and Brodie,
1975). T h e presence of serum prevents extensive breakage of outer segments which
occurs in ordinary Ringer's solution, and W o o d r u f f et al. (1977) have shown that rapid
hydrolysis o f e n d o g e n o u s cyclic GMP occurs when o u t e r segments are disrupted. T h e
calf serum contained negligible amounts of cyclic GMP. (Calibration curves for the
radioimmunoassay [see below], constructed with known a m o u n t s o f cyclic GMP were
similar with and without a d d e d serum.) Each retina was then placed in 500/~1 o f modified
Ringer's solution and slowly agitated for 2 rain to detach r o d o u t e r segments. T h e
resulting suspension o f o u t e r segments and outer segment fragments (containing > 1,000
o u t e r segments/microliter) was used without f u r t h e r purification (see W o o d r u f f et al.,
1977 for discussion of purity). A p p r o x i m a t e l y 80% o f the outer segments were osmotically
intact as d e t e r m i n e d by the fluorescence assay o f Yoshikami et al. (1974). In experiments
where more than one retina was needed, dissections were p e r f o r m e d by more than one
person so that the time between frog sacrifice a n d d e t a c h m e n t o f o u t e r segments from
the retina was fairly constant between experiments. T h e time between sacrifice and
beginning o f the 2-min retinal agitation period was between 3 min and 5 min in all
experiments.
In a typical experiment 50-/~1 portions of the rod o u t e r segment suspension were
withdrawn, exposed to differing conditions o f illumination, o r incubated for different
periods o f time, and then mixed with 90 /~1 of 9% perchloric acid (PCA) to quench
e n d o g e n o u s reactions. Cyclic GMP content was assayed as described below. Details for
each e x p e r i m e n t are given in the figure legends.
In the time-course experiments o f Figs. 2, and 5-7, a series o f silicone-treated Pasteur
pipettes, each containing PCA, was m o u n t e d above a series of rod o u t e r segment
samples. Acid was sequentially ejected onto the samples by squeezing a bulb attached to
the top of each pipet. In the r a p i d time-course e x p e r i m e n t s o f Figs. 3 and 4, acid
quenching o f the series o f o u t e r segment samples was a u t o m a t e d to increase time
resolution. Each Pasteur pipet contained 100/~1 o f PCA m o u n t e d above a 20-/~1 portion
o f rod o u t e r segment suspension so that enzymic reactions could be quenched more
quickly. Acid was ejected from each pipet as a m o t o r driven wheel was rolled across
r u b b e r tubing attached to the pipet. T h e m o t o r (115 V, DC Bodine T y p e f r a m e , 1/s h p ,
1,725 r p m , Bodine Electric Co., Chicago, Ill.) was connected to the wheel by a wafer
magnetic clutch (PIC Design Div. o f Benrus Corp., Ridgefield, Conn.). 12 individual
Published May 1, 1979
632
T H E JOURNAL o r
GENERAL PHYSIOLOGY 9 VOLUME 7 3 9 1 9 7 9
RESULTS
The Amplitude of the Light-Dependent Cyclic GMP Decrease Is a Function of
Time after Isolating Rod Outer Segments from the Retina
T h e cyclic G M P c o n t e n t o f d a r k o u t e r s e g m e n t s slowly d e c r e a s e s a f t e r t h e y a r e
i s o l a t e d f r o m t h e r e t i n a (Fig. 1 a ) , a n d t h e m a x i m u m o b t a i n a b l e l i g h t - i n d u c e d
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outer segment samples could be quenched within 750-1,500 ms. Microswitches were used
to signal the actual time between quencing the first and twelfth samples and to activate a
shutter which exposed the samples to illumination between the quenching of" the fifth
and sixth samples. A high speed movie camera (Lo Cam, Redlake Corp., Photo
I n s t r u m e n t DIP., Campbell, Calif.) adjusted to 100 frames/s, was used to p h o t o g r a p h 12
calibration runs; three runs each at four different m o t o r speeds. T h e ejection o f PCA
from the Pasteur pipets onto the samples was linear with time in all experiments, and the
e r r o r in estimating the time at which each sample was physically contacted by the acid
was <5 ms. T h e time required for mixing acid and sample was also d e t e r m i n e d d u r i n g
the p h o t o g r a p h i c calibration by monitoring a p H color change (bromopheno] blue, p H
range = 3.0-3.6; deep blue ~ clear). Mixing was complete within 52 -+ 18 ms (mean +SD) of initial exposure to the acid. Occasionally (3 cases in 128 determinations) the acid
inverted with the sample and mixing was very s l o w - l o n g e r than 120 ms. These samples
were not used in calculating the mixing time.
T o estimate the additional time required for acid to penetrate outer segment
m e m b r a n e s after mixing, a stopped flow spectrophotometer was used ( D u r r u m Model
D-110, modified to reduce t e m p e r a t u r e artifacts, D u r r u m I n s t r u m e n t Corp., Sunnyvale,
Calif.). T h e device mixed o u t e r segments and acid in 1 ms a n d then monitored an optical
density increase at 515 nm caused by an increase in light scattering. (No such increase
was observed when outer segments were mixed with Ringer's solution instead of
perchloric acid.) T h e increase in density had a half-time of 60 + 13 ms (mean -+ SD). T h e
time for mixing and acid penetration o f the rod outer segments was also estimated by
visually monitoring the acid-induced color change accompanying the denaturation of
r h o d o p s i n (red to orange-yellow). T h e time for the color change was c o m p a r e d with
flashes o f light of various durations. T h e color change was completed within 125-250 ms.
Cyclic GMP levels were assayed as described previously ( W o o d r u f f et al., 1977) using
the radioimmunoassay technique o f Steiner et al. (1972) as modified by Weinryb et al.
(1972). C o m p o n e n t s of the assay were purchased from Collaborative Research Inc.,
Waltham, Mass. Phosphodiesterase activity was assayed using the two-step p r o c e d u r e
outlined by T h o m p s o n et al., (1974) and K e m p and H u a n g (1974). [JH]Cyclic GMP (108
d p m / m m o l , New England Nuclear, Boston, Mass.) was a d d e d to each 50op.l sample o f
rod outer segment suspension. After an a p p r o p r i a t e incubation time and light exposure,
samples were placed in a hot sand bath (-100~
for 1 min to stop enzymic reactions.
Samples were b r o u g h t to r o o m t e m p e r a t u r e , and 200/zl Crotalus atrox venom (2 mg/ml,
50 mM T r i z m a base buffer, p H 7.5) was a d d e d . Samples were incubated with venom for
30 rain at 37~ to convert 5'GMP to guanosine. This reaction was also stopped by placing
the samples in hot sand for 1 min. Samples were then passed t h r o u g h an anion exchange
column (DEAE-Sephadex, A-25, 7.5 • 0.5 cm, Pharmacia Fine Chemicals, Piscataway,
N.J.) equilibrated with 50 mM T r i z m a base, p H 7.5. T h e radioactivity from the first 5 ml
of eluant ([3H]guanosine) was d e t e r m i n e d by liquid scintillation counting (Mark II,
Searle Radiographics Inc., Des Plaines, Ill.). Unreacted cyclic GMP a n d 5'GMP do not
elute from the column until the ninth ml. Recovery from exchange columns was better
than 90%. All chemicals were obtained from Sigma Chemical Co., St. Louis, Mo.
Techniques for illumination o f outer segments a n d for measuring r h o d o p s i n content
are described in an earlier p a p e r (Brodie and Bownds, 1976).
Published May 1, 1979
WOODRUVVANDBOWNDS Light-Induced Decrease in Cyclic GMP: Characteristics
633
decrease b e c o m e s p r o p o r t i o n a l l y less (Fig. 1 b). In the e x p e r i m e n t o f Fig. i a the
c o n c e n t r a t i o n o f cyclic G M P d e c a y e d in the d a r k f r o m 0.0125 mol c G M P / m o l
r h o d o p s i n to 0.0078 tool c G M P / m o l r h o d o p s i n between 6 a n d 30 m i n a f t e r
isolating the o u t e r s e g m e n t s . T h i s slow decrease in cyclic G M P is a c o m m o n
f e a t u r e o f these p r e p a r a t i o n s a n d was n o t e d previously ( W o o d r u f f et al., 1977).
T h e c o n c e n t r a t i o n o f cyclic G M P a f t e r 5-10 min o f isolation varies f r o m
•Q
1.5
x
t-
light
o
o
1,0
E
u
0.5
I
I
I
I
I
I
I
b
r
n
SO
o
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40
~
~
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30
Q.
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_J~
I
I
I
I
I
I
5
I0
18
20
25
30
35
Minutes ofter isoloting outer segments from retino
FIGURE 1. The light-induced decrease in cyclic GMP becomes smaller with time
after detaching rod outer segments from the retina. (a) Outer segments from two
retinas were combined in a total of 2.0 ml modified Ringer's solution (see Methods).
At 6, 13, 20, and 30 min three 50-t~l portions from the dark outer segment
suspension were quenched by addition of 90 ~1 of 9% perchloric acid (O). At 9, 14,
and 22 min, a 200-/~1 portion of the outer segment suspension was exposed to
saturating illumination that bleached 5.0 x 105 rhodopsin molecules/outer segment
per second for 30 s and then three 50-/~1 portions from the illuminated suspension
were quenched as above (O). (A maximum decrease in cyclic GMP is obtained in 30
s; see Fig. 2.) Each data point represents a mean cyclic GMP level - SE of the
triplicate samples. (b) Averages of the percent decreases taken from 14 further
experiments in which maximal cyclic GMP responses to saturating illumination
were determined during this time period.
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._u
Published May 1, 1979
634
T H E .JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 7 3 9 1 9 7 9
The Light-Induced Decrease in Cyclic GMP Is Rapid
Fig. 2 illustrates a time-course for the light-induced decrease in cyclic GMP in
response to illumination which bleaches 5.0 x 10s rhodopsin molecules/outer
segment per second. In the experiment shown the level of cyclic GMP was
reduced approximately 50% within 30 s. Half of this decrease had occurred by
one second of illumination. The inset in Fig. 2 illustrates hydrolysis of exogenous [3H]cyclic GMP by the rod outer segment phosphodiesterase which is
activated by light. Activity of the enzyme is given by the slopes of the lines
shown, and is enhanced at least 20-fold by light which bleaches 5.0 x I(P
rhodopsin molecules/outer segment per second. This light activation of rod
outer segment phosphodiesterase has been documented by several laboratories
(Miki et al., 1973; Keirns, et al., 1975; Krishna et al., 1976; Goridis and Weller,
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preparation to preparation but is generally between 0.010 and 0.020 tool cGMP/
tool rhodopsin. If one assumes that there are 3 • 109 rhodopsin molecules in
each outer segment (Liebman and Entine, 1968) this corresponds to 3-6 • 107
cyclic GMP molecules per outer segment.
Illuminating the outer segments with light of saturating intensity for the cyclic
GMP decrease (5.0 • I(P rhodopsin molecules bleached/outer segment per
second [Woodruff et al., 1977]; and see Fig. 7) at 9, 14, and 22 min of isolation
(Fig. 1 a) decreased cyclic GMP levels 27, 25, and 15%, respectively. In Fig. 1 b
the percent decrease in cyclic GMP averaged from many experiments is
represented as a function of the time after isolating the outer segments from
the retina. The average light-dependent decrease is approximately half as large
at 22 min (20% decrease) as it is at 7 min (42% decrease). No light-dependent
decrease in cyclic GMP was obtained 90 min after the outer segments were
removed from the retina (two determinations, data not shown).
It was considered desirable to determine the effects of illumination as soon as
possible after outer segments were isolated from the retina so that large, easily
measurable responses could be obtained, and any effects of outer segment
deterioration that might occur with time would be minimized. However,
experimental manipulations made it difficult to routinely examine light effects
earlier than 9 min after isolation. In the following experiments (with noted
exceptions) the effects of illumination on rod outer segment cyclic GMP levels
were determined between 9 and 15 min after isolating the outer segments from
the retina.
Even with saturating conditions of illumination, cyclic GMP levels were never
observed to fall more than 50% (see also Woodruff et al., 1977), and thus there
appears to be a "light-sensitive pool" of cyclic GMP containing approximately 2
• l0 T molecules per outer segment. When outer segments are lysed by either
freezing and thawing, homogenization, or hypoosmotic shock, at least 80% of
the total cyclic GMP is degraded rapidly (Woodruff et al., 1977). This suggests
that the cyclic GMP which is not degraded in light exposed outer segments is
inaccessible to the phosphodiesterase. The location of cyclic GMP within the
outer segment and its physical state (soluble or bound) is not known. Phosphodiesterase presumably is restricted to outer segment membranes (Miki et al.,
1975).
Published May 1, 1979
WOODRUFFANDBOWNDS Light-Induced Decrease in Cyclic GMP: Characteristics
635
1976), and is probably the major cause o f the cyclic GMP decrease shown in Fig.
2 (see below).
Several experiments similar to the ones shown in Fig. 2 indicated that the
decrease in cyclic GMP and the activation of phosphodiesterase occur with little
.
9
--
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,~ ~
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/ /
2
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0
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o
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0
0
0
~
0
0
0
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30
I
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0
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20
30
40
50
Seconds
from 1he o n s e t
I
r
I
I
I
70
I10
eo
I
of i l l u m i n o t i o n
FIGURE 2. The light-induced decrease in cyclic GMP and light activation of rod
outer segment phosphodiesterase. Rod outer segments from two retinas were
divided into 50-/~1 portions. After the first three samples had been acid quenched
(O), the remaining portions were sequentially acid quenched (see Methods) while
exposed to continuous illumination that bleached 5.0 x 105 rhodopsin molecules/
outer segment per second (9 The onset of illumination, denoted by a step at the
bottom of the figure, occurred 410 min after isolating the outer segments from
the retinas. Four samples were masked from the light to serve as dark controls.
The average of duplicate points for the controls taken at 37 and 75 s after the onset
of illumination were 98.7% and 97.5% of the average dark cyclic GMP levels,
respectively (data not shown). The line through the data points is drawn by hand.
Inset. The rate of hydrolysis of exogenous cyclic GMP by phosphodiesterase is
enhanced 20-fold by illumination bleaching 5.0 x 10~ rhodopsin molecules/outer
segment per second. Outer segments from two retinas were combined in modified
Ringer's solution with 2 mM cyclic GMP ([aH]cGMP; 100,000 cpm). No exogenous
GTP or ATP was present. The radiotracer was added 5 min, and illumination was
presented 8 min (arrow) after isolating the outer segments from the retinas.
Reactions were stopped by exposing portions of the outer segment suspensions to
a hot sand bath, and the amount of cyclic GMP hydrolyzed was determined as
described in Methods.
delay o n illumination. T o d e t e r m i n e how rapidly cyclic GMP levels decrease,
the a u t o m a t e d q u e n c h i n g device described in Methods was used. Fig. 3 shows a
result obtained using this machine. W h e n o u t e r segments were illuminated with
saturating light intensity (5.0 x 105 r h o d o p s i n molecules b l e a c h e d / o u t e r segment
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50
Published May 1, 1979
636
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
" VOLUME
73
-
1979
per second), the level of cyclic GMP was reduced 30% within 100 ms o f
illumination. Notice, however, that the concentration o f cyclic GMP apparently
started to decay before the onset o f illumination. Evidently the effect o f the acid
on the sample was sufficiently delayed that endogenous reactions were not fully
quenched by the time light was turned on. This means that the time between
the onset o f illumination and the initiation o f the cyclic GMP decrease (latency)
cannot be accurately determined with this acid-quenching technique. However,
if one makes the reasonable assumption that the mixing and quenching time is
constant for each sample, the data still yield accurately the rate at which cyclic
GMP disappears. T h e half-time for this disappearance is 100-150 ms. T h e data
lOS
o
fit.
l[
I00
95
90
.o
80
(0
75
0
i
70
o
65
60o
=o
55
=,
1
-400
I
-300
I
-zoo
I
-Ioo
I
o
I
ioo
I
zoo
Milliseconds from the
I
300
onset
of
I
400
I
soo
I
soo
I
700
illumination
FIGURE 3. The light-dependent decrease in cyclic GMP measured in a rapid dinecourse experiment. 12 outer segment samples were acid-quenched in succession
within 1.05 s (see Methods). Saturating illumination (bleaching 5.0 x 10s rhodopsin
molecules/outer segment per second) was introduced after the fifth sample had
been exposed to the perchloric acid. See text for details.
can be adjusted, as in Fig. 4, to reflect the fact that cyclic GMP levels actually fall
after the onset o f illumination. In 13 experiments similar to Fig. 3 the rate of
decay was extrapolated, using linear regression analysis, to determine that the
apparent beginning o f the decrease occurred ---145 ms before the onset o f
illumination. It was f u r t h e r determined for these experiments that the average
time between the last point obviously not affected by illumination and the onset
o f illumination was 160 -+ 20 ms. If one adds this time (160 ms) to the individual
datum points, thereby shifting the response to the right relative to the onset o f
illumination, the decrease in cyclic GMP can be represented as occurring after,
rather than before, illumination (Fig. 4).
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85
o
Published May 1, 1979
WOODRUrVANDBOWNDS Light-Induced Decrease in Cyclic GMP: Characteristics
637
T o estimate the actual latency o f the cyclic GMP decrease o n e must know the
total time r e q u i r e d f o r mixing acid with the o u t e r segments ( 2 5 0 ms, see
Methods), a n d for q u e n c h i n g e n d o g e n o u s reactions. T h e q u e n c h i n g time is
probably d e t e r m i n e d by the time r e q u i r e d for acid to penetrate the o u t e r
105
~100
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I
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I
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I
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I
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SO0
illuminotion
FIGURE 4. Rapid decrease in rod outer segment cyclic GMP levels measured at
three different light intensities. 25 experiments were performed using the fast
time-course device. 13 experiments used light intensity which was saturating for
the cyclic GMP decrease (5.0 x 10~ rhodopsins bleached/outer segment per second
[O]), 6 experiments used light intensity two orders of magnitude below saturation
(5.0 x 10a rhodopsins bleached/outer segment per second [9
and another 6 used
illumination two orders of magnitude above saturation (1.2 x 107 rhodopsins
bleached/outer segment per second [X]). After adjusting each individual datum
point by 160 ms, as described in the text, all of the data points were entered on a
single graph and the points within discrete time intervals were averaged. The
points shown in the figure represent the mean _+ the standard error of the mean
for all individual data points that fell within an interval. For the saturating intensity
(e), enough data was available so that 50-ms intervals (between -300 ms and 300
ms) could be used for averaging. For the other intensities 100-ms intervals were
used. Curves were drawn by hand. The latency and the rate of the cyclic GMP
decrease were approximately similar at all intensities.
segment membranes (t]/~ m; 60 ms, see Methods) and the nature of the reactions
q u e n c h e d . In soluble systems acid o r alkali q u e n c h i n g requires < 1 - 3 ms
( B a r m a n and G u t f r e u n d , 1964). L i e b m a n et al. (1978) have recently used a rapid
kinetic assay to d e m o n s t r a t e that phosphodiesterase activity becomes maximal
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arlu
tD
Published May 1, 1979
638
T H E JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 7 3 " 1 9 7 9
The Light-Induced Decrease in Cyclic GMP Reverses When Illumination Ceases
The data of Figs. 5 and 6 demonstrate that cyclic GMP levels return to near
their dark-adapted values after brief illumination at various light intensities.
The results of these experiments, together with 16 others not shown, are
summarized in Table I. If outer segments are exposed for 1 s or less to light
bleaching between 5.0 x 102 and 5.0 x 105 rhodopsin molecules/outer segment
per second, recovery of cyclic GMP levels is essentially complete (Figs. 5, 6, and
Table I). After the most intense illumination used (1.2 x 107 rhodopsin
molecules bleached/outer segment per second, delivered for 2 ms to 1 s) the rate
of recovery was slowed, but cyclic GMP levels still recovered to within 6% of
unilluminated control levels (Table I). (The extent of recovery was determined
in each experiment after correcting for decay of cyclic GMP levels in an
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within milliseconds after a bright flash. It seems likely then that cyclic GMP
levels would begin to decrease within milliseconds after light absorption.
The results of 25 fast time-course experiments, in which the data were
adjusted as just described, are presented in Fig. 4. The outer segments were
exposed to three different light intensities. With a light that was just saturating
for the response (5.0 • 105 rhodopsin molecules bleached/outer segment per
second; Q) cyclic GMP was reduced by 35% from its dark-adapted concentration
with a half-time of 125 ms. Light two orders of magnitude brighter (X) did not
significantly increase the absolute latency, rate, or extent of cyclic GMP decay.
Exposure to intermediate levels of illumination (9 caused a smaller decrease
(10-15% compared to 30-35% for saturating intensity), but the latency of the
decrease did not seem to be significantly longer. (This intermediate level of
illumination causes a half-maximal permeability change in isolated outer segments [Brodie and Bownds, 1976; Woodruff et al., 1977]).
The total light induced decrease in cyclic GMP occurs in two phases. The
rapid decrease shown in Figs. 3 and 4 is followed by a slower decay that occurs
over the next few seconds (shown in Fig. 2; and in Fig. 3 of Woodruff et al.,
1977). In two of the experiments in Fig. 4 (with light bleaching 5.0 • I(P
rhodopsin molecules/outer segment per second) 10 additional data points were
taken beyond 800 ms of illumination to illustrate this behavior. Within 1 s of
illumination, cyclic GMP levels decreased by 30%, and within the next 20 s an
additional fall of 10-15% completed the light-induced change (data not shown).
Given that there are between 3 • 107 and 6 • 107 cyclic GMP molecules/outer
segment and 3 • 109 molecules of rhodopsin, one can calculate that the rate at
which cyclic GMP molecules disappear during the rapid decrease shown in Fig.
4 is between 4 • 104 and 8 • 104 molecules/outer segment per millisecond.
Assuming Michaelis-Menten kinetics, a cyclic GMP concentration of 10-5 M, a
K m of 0.07 mM (Miki et al., 1975), and a Vmax of 20 mol cyclic GMP/mol
rhodopsin per minute (initial velocity calculated from Fig. 2, inset), the fully
light-activated phosphodiesterase might hydrolyze between 1.2 • 105 and 2,2 •
105 molecules of cyclic GMP/outer segment per millisecond. Thus, phosphodiesterase activation alone is probably sufficient to produce the rapid decrease
shown in Fig. 4, if the rate of cyclic GMP synthesis is unaltered by illumination,
and if PDE activation is as rapid as reported by Liebman et al. (1978).
Published May 1, 1979
WOODRUFF AND BOWNDS
639
Light-Induced Decrease in Cyclic GMP: Characteristics
u n i l l u m i n a t e d control. T h e average decay for this control was 8-9% d u r i n g the
80-s p e r i o d cyclic G M P levels were followed.) Recovery a p p e a r e d to be c o m p r o mised not by the intensity o f the illumination but by its d u r a t i o n . For example,
after 10 s o f illumination bleaching 5.0 x liP r h o d o p s i n molecules/outer segment
p e r second, cyclic G M P r e c o v e r e d to only 83% o f the d a r k - i n c u b a t e d control,
but after illumination which bleached 2.4 times m o r e r h o d o p s i n in 1 s, 96%
recovery was obtained (Table I). With longer light e x p o s u r e s a longer delay
l
I
I
I
l
I
I|
!
I
I
I
I
,.
I
I
I 5 x io0, rhod./o.s., S
1
I
5xlO r h o d . ~ s . . s
o
I00
o
o
_? 9 0
o
o
O.
~E 80
(,9
o
o
o
o
o
~
o
o
a
70
~" 6o
"~ 5o
_n
9
n
I
I
I
I
I
I
i
14
I
I
5x I0 rhodJo.s,-s
i
i
I
1
Is
I
I
5xlO r h o d J o s - s
Ol I00
o
ID
> 9O
0
f
"~ eo
o
~
7o
lID
o
1~. 6 o
SO
i
o
i
,o
|
2o
i
3o
i
40
i
so
Seconds
I
6o
l
70
from
i
80
the
|
o
onset
l
,o
of
[
zo
I
3o
I
,to
|
~o
I
6o
I
70
i
eo
illuminotion
FIGURE .5. The light-dependent decrease in cyclic GMP as a funcdon of light
intensity. ]n four separate experiments rod outer segments were exposed for 1 s to
light bleaching 5.0 x 102, 5.0 • 10a, 5.0 x 10a, or 5.0 • 10~ rhodopsin molecules/
outer segment per second. The average dark cyclic GMP level was determined for
each individual experiment. As light intensity increased the amplitude of the cyclic
GMP decrease increased (see also Fig. 7). Similar data for each intensity was
obtained in three separate experiments.
between the cessation o f illumination a n d the start o f the cyclic G M P recovery
was also observed. After e x p o s u r e to 2 ms o r 125 ms o f illumination, cyclic GMP
recovery was initiated in less than 10 s, but after a 1-s or 10-s e x p o s u r e , 10-20 s
were r e q u i r e d (data not shown).
The Amplitude of the Cyclic GMP Decrease Is a Function of the Intensity and
Duration of lUumination
T h e data f r o m e x p e r i m e n t s such as those shown in Figs. 5 and 6 yield
i n f o r m a t i o n o n the a m p l i t u d e o f the cyclic G M P decrease u n d e r varying
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0
Published May 1, 1979
640
THE
JOURNAL
or
GENERAL
PHYSIOLOGY
' VOLUME
73 9 1979
conditions o f illumination, a n d can be used to c o n f i r m a n d e x t e n d the findings
o f W o o d r u f f et al. (1977). In that p a p e r it was s h o w n that the a m p l i t u d e o f the
cyclic G M P decrease increased with the l o g a r i t h m o f light intensity at levels
which bleach between 5.0 • 102 and 5.0 x 105 r h o d o p s i n molecules/outer
s e g m e n t p e r second. T o e x a m i n e this relationship f u r t h e r o u t e r s e g m e n t s were
e x p o s e d for 2 or 125 ms o r 1, 10, a n d 60 s to illumination bleaching between 5.0
• 102 a n d 1.2 • 107 r h o d o p s i n molecules/outer s e g m e n t p e r second, a n d the
I
I
I
I
o
I
I
I
I
I
I
0.002 S
I
t
I
I0
I
9
I
o
I
I
0.125 S
I00
eel
-~ 90
n
o
o
%
-
o
o
80
~9
._u
o
o
7o
/
0
50
I
I
t
I
t
I$
I
t
I
lOS
i~,
I00
o
90
"6
"E
80
o
7o
oo,~
2
t
o
6O
fl
I
50
0
I
20
~
40
50
Seconds
60
70
8
"1
,~ 2'0 io ,~
5~ 6~ ;o 9'0
from the onset of illumination
FIGURE 6. The light-induced decrease in outer segment cyclic GMP levels as a
function of time of exposure to illumination. In four separate experiments rod
outer segments were exposed to illumination bleaching 5.0 • 10s rhodopsin
molecules/outer segment per second for 2 or 125 ms, or 1 or 10 s. The outer
segments were prepared and extracted as in Fig. 2. Similar data for each condition
was obtained in at least three separate experiments.
m a x i m a l cyclic G M P decreases were d e t e r m i n e d . T h e a m p l i t u d e s o f the responses f o r each condition are indicated in Fig. 7.
T h e a m p l i t u d e o f the cyclic G M P decrease is similar w h e n m e a s u r e d after 1,
10, or 60 s o f illumination a n d is g r a d e d with the l o g a r i t h m o f light intensity
o v e r the r a n g e between 5.0 x 102 and 5.0 x 104 r h o d o p s i n molecules bleached/
o u t e r s e g m e n t p e r second. T h i s is consistent with the data a n d discussion in
Figs. 2 a n d 4, a n d indicates that cyclic G M P c o n c e n t r a t i o n in o u t e r s e g m e n t s
falls to a value characteristic for a given level o f illumination within several
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~ 6o
Published May 1, 1979
WOODRUFF AND BOWNDS Light-Induced Decrease in Cyclic G M P : Characteristics
641
seconds after the onset of illumination, and then remains at that level even
though more rhodopsin is being bleached as illumination continues. For
illumination lasting 1 s or longer, cyclic GMP levels must then be determined by
the rate of rhodopsin bleaching, and not the absolute number of rhodopsin
molecules bleached. Cyclic GMP is being continually synthesized and degraded
in these isolated outer segments, and it would appear that light acts by shifting
the balance between synthesis and degradation so that cyclic GMP level comes to
a new steady value (see Discussion).
Illumination which lasts for 2 or 125 ms does not trigger the maximal cyclic
GMP change possible for a given intensity at even the brightest intensity used
(1.2 x 107 rhodopsin molecules bleached/outer segment per second). Even
though 2 ms of this bright intensity bleaches approximately the same number of
rhodopsin molecules as a 1-s exposure to an intensity of 5.0 x 104 rhodopsin
TABLE
RECOVERY
Light intensity
AFTER
Extent of recovery
Rhodota'in radeodes bleached/
outer segmentper second
% of dark-~laOted control
BRIEF
Rate of recovery
% increase/second
2 ms
5.0xlO n
1.2 X 107
110+--3
98+-- 1
1.1
0.5
125 m s
5 . 0 x 105
1.2x 10'
100---2
94•
0.9
0.4
ls
5.0x 10a
5 . 0 x 10s
1.2 x 10'
100__.4
95---2
96--- 1
0.7
0.6
0.2
10 s
5 . 0 x 10a
5 . 0 x 105
92*
8 3 +- 1
0.6
0.4
In a n u m b e r o f e x p e r i m e n t s s u c h as those s h o w n in Figs. 5 a n d 6, t h e e x t e n t o f
recovery o f cyclic G M P was d e t e r m i n e d by c o m p a r i s o n with d a r k - a d a p t e d
controls (see text). T h e rates o f recovery are estimates a n d were d e t e r m i n e d by
m e a s u r i n g t h e slopes o f t h e curves after cyclic G M P started to r e t u r n towards its
d a r k level. Each value is t h e average f r o m t h r e e d e t e r m i n a t i o n s (+-SE).
* Value is t h e average o f two d e t e r m i n a t i o n s .
molecules bleached/outer segment per second, the cyclic GMP decrease caused
by the briefer illumination is significantly lower (see Fig. 7). For short light
exposure the amplitude of the cyclic GMP decrease is a function of both the rate
of rhodopsin molecule bleaching and the duration of exposure. This suggests
that some component important in setting the maximum amplitude of the cyclic
GMP decrease is expressed (or perhaps reaches a critical concentration) only if
photoexcited rhodopsin molecules, or a short-lived rhodopsin intermediate, are
present for a period of at least 1 s.
The first few photons captured in completely dark-adapted photoreceptors
each cause 0.1-1.0% of the total possible permeability change (see Baylor et al.,
1974; Schwartz, 1975; Fain, 1976). From the information in Fig. 7 one can
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Time of light exposure
I
OF CYCLIC GMP LEVELS
ILLUMINATION
Published May 1, 1979
642
THE JOURNAL
OF GENERAL
PHYSIOLOGY
' VOLUME
73 9 1979
estimate h o w m a n y cyclic G M P molecules d i s a p p e a r f r o m the cyclic G M P pool
in a n o u t e r s e g m e n t f o r each r h o d o p s i n m o l e c u l e b l e a c h e d . T h e largest value
f o r this ratio is o b t a i n e d f o r s h o r t e x p o s u r e to d i m light, the smallest f o r l o n g
e x p o s u r e to b r i g h t light. A p p r o x i m a t e l y 3 • 104 a n d 5 • 10 4 m o l e c u l e s o f cyclic
!
45
~ 40
u
U
"O
0
0
O
0
0
O
"O
I0
tD
,
L
IO s
,
i
I0"
,
i
I0~
,
i
I 0r
,
~j
I
Li~iht Intensity (rhodopsin molecules bleached/outer segment-s)
T h e amplitude of the cyclic GMP decrease as a function of light
FIGURE
7.
intensity and exposure duration. Outer segments were exposed for 2 ms (El), 125
ms (A), 1 s (O), 10 s (Q), or 60 s (B) to illumination bleaching 5.0 x los, 5.0 • l0 s,
5.0 x 104, 5.0 x I(P, or 1.2 x 107 rhodopsin molecules/outer segment per second.
T h e maximum decrease in each experiment (which occurred 2-20 s after the onset
of illumination, depending on the duration of illumination) was determined using
the three to five individual data points from the time-course (see Fig. 5 and 6)
which gave the maximum difference between light and dark. The points shown
are an average from at least three separate experiments (with the exception that
points for the 60-s exposure at 5.0 x 105 and 1.2 x l0 T rhodopsin molecules
bleached/outer segment per second and the 10-s exposure at 5.0 x 105 rhodopsin
"nolecules bleached/outer segment per second are the average of two determinations). Standard errors for 1-s and 10-s response curves are not indicated to permit
the curves to be seen more clearly; they are similar to the errors shown for the 60s curve. The response is larger at high light intensities, and is nearly complete after
a 1-s exposure. Illumination for 125 or 2 ms at even the highest intensity does not
give a maximal cyclic GMP decrease.
G M P d i s a p p e a r p e r r h o d o p s i n m o l e c u l e b l e a c h e d after 2 ms o f light b l e a c h i n g
5.0 x 104 r h o d o p s i n m o l e c u l e s / o u t e r s e g m e n t p e r s e c o n d , a n d 125 ms o f light
b l e a c h i n g 5.0 x 102 r h o d o p s i n m o l e c u l e s / o u t e r s e g m e n t p e r s e c o n d , respectively. T h e ratio r a p i d l y d e c r e a s e s f r o m this value as m o r e r h o d o p s i n m o l e c u l e s
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E 2Q
Published May 1, 1979
643
WOODRUFFANDBOWNDS Light-Induced Decrease in Cyclic GMP: Characteristics
are bleached. T o obtain an estimate o f the n u m b e r o f cyclic G M P molecules
which d i s a p p e a r at even lower light intensities, one can extrapolate f r o m the
data o f Fig. 7 g r a p h e d as shown in Fig. 8. Using logarithmic coordinates the
decrease in the n u m b e r o f cyclic GMP molecules per r h o d o p s i n molecule
bleached is plotted as a function o f the n u m b e r o f r h o d o p s i n molecules bleached
per o u t e r segment. With light e x p o s u r e s that bleach > 10~ r h o d o p s i n molecules/
o u t e r segment, the slope o f the curve is - 1 . 0 - 0.01 (mean -+ SE; solid line)
p r e s u m a b l y because the cyclic GMP decrease has saturated and is not influenced
~g
\x
E
x
,g
x
. . . . . . . . . . . . . . . . ~12,,
"-O
"qx
10
..~ i(?
E
O
,
I
I0'
i
I
I0'
Rhodopsin
i I
i.l
IO s
molecules
I l
I0'
L I
I0 a
bleoched/~uter
10'
i
I
10'
i I
I
~0'
segment
FIGURE 8. Decrease in the number of cyclic GMP molecules for each rhodopsin
molecule bleached plotted as a function of the number of rhodopsin molecules
bleached in each outer segment. Points were calculated assuming that dark-adapted
outer segments contain 4.5 x 10* molecules of cyclic GMP. This value was
multiplied by the percent decreases shown in Fig. 7 to obtain the actual number of
cyclic GMP molecules disappearing after differing amounts of rhodopsin bleaching. The solid and dashed lines were determined by linear regression analysis for
the data above and below 105 rhodopsin molecules bleached/outer segment,
respectively. The symbols represent the different exposure durations and are
defined in Fig. 7. (See text for details.)
by additional r h o d o p s i n bleaching. With light e x p o s u r e s that bleach <I(P
r h o d o p s i n molecules/outer segment, the slope is - 1 . 2 2 -- 0.17 (mean -+ SE;
d a s h e d line). I f this is e x t r a p o l a t e d to o n e r h o d o p s i n molecule bleached per
o u t e r segment, - 1 . 5 x 10e molecules o f cyclic GMP, 3.3% o f the total pool o f
o u t e r s e g m e n t cyclic GMP, would disappear after a single p h o t o n is c a p t u r e d .
T h e actual decrease caused by a single p h o t o n absorption is probably between
this e x t r a p o l a t e d value a n d the experimentally derived value o f 3-5 x 104 cyclic
GMP molecules d i s a p p e a r i n g / r h o d o p s i n molecule bleached, which represents
a p p r o x i m a t e l y 0.1% o f the total cyclic GMP.
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-a
|
E
Published May 1, 1979
644
THE JOURNAL
OF GENERAL
PHYSIOLOGY
' VOLUME
73 "
1979
The Decrease in Cyclic GMP Induced by Light Superimposed on Background
Illumination Is Reduced by an Amount Proportional to the Intensity of the
Background Light
Removal of Calcium Enhances Cyclic GMP Levels in Dark-Incubated Outer
Segments
In several electrophysiological studies it has been noted that procedures expected to lower calcium ion concentration in rod outer segments depolarize the
membrane of the dark-adapted rod (presumably by increasing plasma membrane permeability) (Lipton et al., 1977 a; Brown et al., 1977). All of the
experiments in this report and in our previous papers (Bownds and Brodie,
1975; Brodie and Bownds, 1976; Woodruff et al., 1977) have in fact been carried
out in a modified Ringer's solution supplemented with 3 mM EGTA to bring
the free calcium concentration to 10-s M. These conditions increase the
permeability of isolated outer segments as well as the magnitude of the lightinduced permeability decrease (Bownds and Brodie, 1975) and have made the
experiments which correlate enzyme activities or cyclic GMP levels with the
permeability of isolated outer segments technically more feasible (Brodie and
Bownds, 1976; W o o d r u f f et al., 1977).
Lowering the calcium concentration increases not only the permeability of
isolated outer segments (Brodie and Bownds, 1975) but also their cyclic GMP
levels. Rod outer segments prepared in Ringer's solution containing 10-3 M
Ca ++ contained an average of 1.04 - 0.06 x 10-2 mol cGMP/mol rhodopsin
(•
n = 3), whereas those in Ringer's solution containing - 1 0 -8 M Ca ++ (i.e.,
3 mM EGTA) averaged 1.53 - 0.14 • 10-2 mol cGMP/mol rhodopsin. Alternatively, if rod outer segments were prepared in a low calcium Ringer's solution
(3 mM EGTA) and exposed to 4 • 10-3 M CaCI2 (bringing free Ca ++ to 10-3 M),
cyclic GMP levels decreased by 27.5 • 2.3% (mean • SE, n = 15; for example,
Downloaded from on June 17, 2017
In living photoreceptors the effect of dim background illumination is to make
the receptor less responsive to light superimposed on that background (for
example, see Fain, 1976). Thus, the effects of introducing steps of illumination
were examined in the outer segment preparation. The data presented in Table
II demonstrate that dim illumination (bleaching 5.0 • 101 rhodopsin molecules/
outer segment per second) has no influence on the final level to which cyclic
GMP is reduced by either an intermediate light intensity (bleaching 5.0 • 103
rhodopsin molecules/outer segment per second) or a saturating light intensity
(bleaching 5.0 • 105 rhodopsin molecules/outer segment per second). The same
result is obtained when the saturating illumination is superimposed on brighter
background lights (Table II).
Because the first step of illumination (background light) partially decreased
the level of cyclic GMP without affecting the final level to which cyclic GMP was
decreased by the second step of illumination (test light), the cyclic GMP response
to the test light was reduced compared to the response in outer segments not
exposed to the background light. The magnitude of this reduction is proportional to the magnitude of the cyclic GMP decrease induced by the background
light.
Published May 1, 1979
645
WOODRUFFAND BOWNDS Light-Induced Decrease in Cyclic GMP: Characteristics
see F i g . 9). It w o u l d a p p e a r t h a t cyclic G M P c o n c e n t r a t i o n is i n f l u e n c e d by
c h a n g e s in e x t e r n a l c a l c i u m in t h e c o n c e n t r a t i o n r a n g e b e t w e e n 10 -8 a n d 10 -5
M, b e c a u s e t h e a d d i t i o n o f 10 -3 M CaCI2 to o u t e r s e g m e n t s p r e p a r e d in R i n g e r ' s
s o l u t i o n c o n t a i n i n g 10 -4 M C a ++ d i d n o t i n f l u e n c e cyclic G M P levels. ( C a l c i u m
c o n c e n t r a t i o n in t h e s e r u m - R i n g e r ' s s o l u t i o n c o n t a i n i n g n o a d d e d E G T A was
10 -4 M , a n d t h e cyclic G M P level was 0.95 - 0.05 m o l / m o l r h o d o p s i n . )
O b s e r v a t i o n s s i m i l a r to t h e s e h a v e b e e n r e p o r t e d f o r l i v i n g r e t i n a s by C o h e n et
al. (1977) w h o f o u n d t h a t i n c u b a t i o n in E G T A - c o n t a i n i n g s o l u t i o n s c a u s e s a
l a r g e s t i m u l a t i o n o f cyclic G M P levels ( 1 0 - 2 0 - f o l d ) , w h e r e a s i n c r e a s i n g e x t e r n a l
c a l c i u m levels c a u s e s n o d e p r e s s i o n o f cyclic G M P levels.
TABLE
II
E F F E C T O F B A C K G R O U N D I L L U M I N A T I O N ON T H E
A M P L I T U D E O F CYCLIC GMP DECREASE
Light intensity
Average dark cyclic GMP
Test light
n
level
Rhodot~sin molecules bleached~outer segment per second
-
5.0• 101
-5.0• 10~
--
5.0x 10~
5.0• 102
-
5.0• 10a
5.0• 103
--
5.0• 10a
%
-
100
5.0• 10a
5.0• 103
5.0•
6
4
4
10 ~
2
5.0• 10s
--
2
2
5.0•
n
2
5.0• I(P
--
2
2
5.0•
2
10s
5.0x 105
2
91 ---3
70---6
68---5
51
57
76
47
47
64
57
54
Rod outer segments were exposed for 15 s to background light of indicated
intensities and then for 6--8 s to a test light. The cyclic GMP decreases induced
by the background light alone, the test light alone, and then the background
followed by the test light are shown for various conditions of illumination.
Background illumination does not significantly influence the level to which
cyclic GMP is reduced by the test light. The number of experiments using each
condition of illumination is indicated (n). In each experiment data points were
averaged from triplicate samples.
Fig. 9 d e m o n s t r a t e s f u r t h e r t h a t a l t h o u g h t h e level o f cyclic G M P in d a r k i n c u b a t e d o u t e r s e g m e n t s is i n f l u e n c e d by c a l c i u m c o n c e n t r a t i o n , t h e final level
to w h i c h cyclic G M P is s u p p r e s s e d b y s a t u r a t i n g i l l u m i n a t i o n is n o t . B e t w e e n 10
a n d 30 m i n a f t e r i s o l a t i n g o u t e r s e g m e n t s f r o m t h e r e t i n a , t h e level o f cyclic
G M P in 10 -3 M C a ++ was a b o u t 25% l o w e r t h a n t h e level in l o w - c a l c i u m R i n g e r ' s
solution. When outer segments were illuminated with a saturating light intensity
( b l e a c h i n g 2.2 • 10 e r h o d o p s i n m o l e c u l e s / o u t e r s e g m e n t p e r s e c o n d ) t h e level
o f cyclic G M P was r e d u c e d to a p p r o x i m a t e l y t h e s a m e level (0.5 • 10 -2 m o l
c G M P / m o l r h o d o p s i n ) in b o t h low a n d 10 -3 M C a ++. I n six s e p a r a t e e x p e r i m e n t s
like t h e o n e s h o w n in F i g . 9, cyclic G M P c o n c e n t r a t i o n in t h e p r e s e n c e o f 1 m M
Ca ++ was 96.0 + 1.6% o f t h a t w i t h --10 -s C a ++ a f t e r e x p o s u r e to s a t u r a t i n g
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Background light
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73 9 1979
illumination. Fig. 9 also shows that illumination bleaching 5.0 • 103 r h o d o p s i n
molecules/outer segment per second decreases cyclic GMP to an intermediate
level in low calcium conditions (consistent with Fig. 7), but almost fully decreases
cyclic GMP concentration in 1 mM Ca ++ Ringer's solution. A conclusion d r a w n
from a n u m b e r o f e x p e r i m e n t s at intermediate levels o f illumination (bleaching
5.0 x 102 and 5.0 x 103 r h o d o p s i n molecules/outer segment per second) is that
the effectiveness o f illumination (the n u m b e r o f cyclic GMP molecules which
disappear for each r h o d o p s i n molecule bleached) is not significantly altered by
I mM Co*§
"~o~1"5~~dlO'eark
Ca*+
"~
5x 103rhod/o,s,-s
~
u
~
~/~5 xI0~rhodlos.-s
~-f2.2 x 106rhod/o~s:s
g
2 2 x I06 rhod /o.s.-s
"6
,s
2'0
2'5
3b
,0
~
2'0
~5
3~
'
Minutes after isoloting outer segments from retino
FIGURE 9. Calcium concentration and rod outer segment cyclic GMP levels.
Outer segments i~rom two retinas were combined in the modified Ringer's solution
used in the experiments of Figs. 1-8 (containing 3 mM EGTA, --10 -~ M Ca++). A
portion of this suspension was made 4 mM in CaCi2, so that the free calcium
concentration was 1 raM. The low calcium and 1 mM Ca ++ outer segment
suspensions were each divided into three portions. One was left dark (9 another
was illuminated with light bleaching 5.0 x 10a rhodopsin molecules/outer segment
per second (Q), and the third was illuminated with light bleaching 2.2 • 106
rhodopsin molecules/outer segment per second (x). Each data point represents the
average (_+ standard error) of triplicate samples (50/zl) taken at the times indicated.
Calcium decreased the dark-adapted level (9 of cyclic GMP --25%. Illumination
bleaching 5.0 • 103 rhodopsin/outer segment per second (O) partially decreased
cyclic GMP in the control Ringer's solution, and fully suppressed cyclic GMP in 1
mM Ca ++.
conditions o f low or high calcium concentration. In high calcium the maximal
light-induced decrease in cyclic GMP thus occurs at a lower light intensity (5.0
• 103 r h o d o p s i n molecules bleached/outer segment per second), because in this
condition there is less cyclic GMP in the dark available for hydrolysis on
illumination.
DISCUSSION
T h e main finding o f this work is that cyclic GMP levels in freshly isolated frog
rod o u t e r segments fall very rapidly u p o n illumination and within a few seconds
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-"4
IE
c.9
k
Published May 1, 1979
WOODRUFFANDBOWNDS Light-Induced Decrease in Cyclic GMP: Characteristics
647
1Biernbaum, M., and D. Bownds. Light-activateddecrease of guanosine5'-triphosphate in isolated
frog rod outer segments. Manuscriptin preparation.
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assume a steady value which is a function of the light intensity. It would appear
that the light-dependent changes noted in this paper, at least among the
nucleotides, may be unique to cyclic GMP. Orr et al. (1976) and Cohen et al.
(1978), have shown that cyclic AMP levels are not light sensitive. Biernbaum and
Bownds 1 have found that ATP levels are not changed by illumination, and that
a light-induced decrease in GTP levels is slower than the cyclic GMP decrease.
The light-induced decrease in cyclic GMP occurs rapidly enough and with
sufficiently high stoichiometry to be involved in the transduction process in rod
outer segments. The cyclic GMP decrease probably occurs within milliseconds
of light absorption, and the half-time for the initial rapid phase is 125 ms. The
light-induced hyperpolarization of the amphibian rod has an absolute latency
between 30 and 50 ms, and the time to peak at moderate to bright light
intensities is between 100 and 200 ms (for example, see Fain, 1976). Adaptation
processes may be initiated within 50-100 ms of the onset of illumination (Baylor
and Hodgkin, 1974). The graph of Fig. 8 suggests that the fraction of total cyclic
GMP which disappears for each of the first few rhodopsin molecules bleached is
between 0.1%, measured after bleaching 60-100 rhodopsin molecules, and a
value of 3.3%, obtained by extrapolation to one rhodopsin molecule bleached.
(Experimental error prevented direct determination of the cyclic GMP decrease
caused by <100 photon absorptions.) Because approximately half of the total
cyclic GMP is light-sensitive, between 0.2 and 6.6% of the light-sensitive cyclic
GMP disappears per rhodopsin molecule bleached for each of the first few
photons captured. In fully dark-adapted photoreceptors, 0.1-1.0% of the total
possible permeability suppression is caused by each photon absorption for the
first 10-20 photons captured (see Baylor et al., 1974; Schwartz, 1975; Fain, 1976)
and the absorption of only a few photons/rod per second triggers a light
adaptation which lowers the sensitivity to about one-third of its dark-adapted
value (Baylor and Hodgkin, 1974; Fain, 1976).
Continuous illumination at intermediate intensities (5 x 102 and 5 x 103
rhodopsin molecules bleached/outer segment per second) causes cyclic GMP
concentration to fall to steady levels which are clearly intermediate between
dark levels and the levels caused by saturating illumination (5 x 104 to 5 x 105
rhodopsin molecules bleached/outer segment per second). 90% of the change
occurs over 3-4 log units of light intensity. The permeability of isolated outer
segments (Brodie and Bownds, 1976) and the hyperpolarizing response of
amphibian rod receptors to continuous illumination (Fain, 1976) are graded
with light intensity over this same range. (Most vertebrate rod cells exposed to
continuous illumination increase their response as the light intensity is raised to
4 log units above threshold, with saturation of the response occurring when
about 104 rhodopsin molecules are being bleached/outer segment per second.
For example, see Normann and Werblin, 1974; Kleinschmidt and DoMing,
1975; Fain, 1976.)
In addition to similarities between the behavior of cyclic GMP and the
electrical activity of photoreceptors, there are at least four significant differ-
Published May 1, 1979
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ences. First, short flashes of bright light (10-100 ms in duration) can cause a
saturating photoreceptor response, but illumination lasting ['or up to 1 s is
required for a maximal cyclic GMP decrease to be registered (Fig. 7). Second,
even though the photoreceptor potential change and the cyclic GMP response
saturate at about the same light intensity, the half'maximal response f'or
photoreceptor hyperpolarization occurs when only 10-20 photons have been
absorbed, whereas the h a l f maximal response f'or cyclic GMP occurs when
--1,000 photons have been captured (or -100 photons when calcium concentration has not been buffered to - 1 0 -s M by EGTA addition). The cyclic GMP
response is less sensitive than the potential response by 1-2 orders of magnitude.
Third, dim background illumination desensitizes the rod receptors response to
light but has no desensitizing effect on the cyclic GMP decrease (Table I1).
Finally, the receptor's membrane potential recovers rapidly after 10 s of'
illumination bleaching 5.0 x 105 rhodopsin molecules/outer segment per second,
but cyclic GMP levels recover slowly and incompletely (Fig. 5, Table I).
Some of these differences may derive from a "slowing down" of the endogenous chemistry of outer segments after their detachment from the retina. Even
though isolated outer segments maintain their ability to synthesize and degrade
cyclic GMP, it is clear that degenerative changes are taking place. Levels of"cyclic
GMP, ATP, and GTP decay in dark adapted outer segments (Fig. 1 a, and
Woodruff et al., 1977; Biernbaum and Bowndsl), presumably because crucial
metabolites or cof'actors can no longer be obtained from the inner segment
portion of" the photoreceptor. The response of" cyclic GMP to illumination
becomes less with time after isolating the outer segments from the retina (Fig. 1
b). The failure of cyclic GMP to recover after a lengthy exposure to illumination
also may be symptomatic of degenerative changes. It is because of this deterioration that no attempt has been made to extend to aged preparations the
correlation, noted by Woodruff et al. (1977), between light suppression of cyclic
GMP and permeability. Although Bownds and Brodie (1975) observed light
suppression of outer segment swelling (permeability) 2 h after detachment of
outer segments from the retina, it has been found that in many preparations
this is not observed. Light suppression of swelling often is lost in <1 h. Thus,
the lability of both the permeability mechanism and the light-induced cyclic
GMP decrease has made it impractical to pursue their correlation in outer
segments detached from the retina for more than 60 min.
Further problems arise in comparing the effects of illumination on the
intracellularly recorded rod response with the cyclic GMP measurements on
isolated outer segments in suspension. The electrical responses recorded from
a single rod derive from a pooling of the signals of many adjacent receptors and
thus do not represent only the receptor which is impaled (Schwartz, 1975; Fain
et al., 1975; Copenhagen and Owen, 1976); further, they might involve voltagedependent conductances arising in the inner segment portion of the photoreceptor (Fain et al., 1978). It will be more relevant to compare the chemical
changes which occur in individual outer segments with conductance changes
which occur in individual outer segments. Yau et al. (1977) have recently
introduced techniques for monitoring the membrane current of single rod
Published May 1, 1979
WOODRUFF AND BOWNDS Light-Induced Decrease in Cyclic GMP: Characteristics
649
Abramson, B. P., R. P. Robinson, M. L. Woodruff, and D. Bownds. Light sensitive enzymes
regulating guanosine 3',5'-cyclic monophosphate concentration in frog rod outer segments. Manuscript in preparation.
3 Green, S., and D. Bownds. Unpublished observations.
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outer segments, and appropriate data on conductance changes in the outer
segment plasma membrane should soon be available for comparison with the
light-induced chemical changes. It is important also to emphasize that data in
these experiments were obtained in low-calcium media; the presence of 3 mM
EGTA brought the free calcium concentration to - 1 0 -~ M. Most of the
electrophysiological data has been obtained by using Ringer's solutions containing millimolar levels of calcium.
In these outer segment preparations, cyclic GMP concentration probably is
regulated by a balance between its synthesis and destruction. The ability of the
outer segments to synthesize cyclic GMP is indicated by an increase in cyclic
GMP levels when inhibitors of the degradative enzyme, phosphodiesterase, are
added (Woodruff et al., 1977) and by the observation that cyclic GMP levels
increase after termination of a brief light exposure (Figs. 5 and 6). Cyclic GMP
concentration is clearly "buffered", for it remains fairly constant even when
levels of its precursor GTP are changed (Biernbaum and Bowndsl). The
mechanism which permits cyclic GMP concentration to stabilize at intermediate
levels of illumination then probably involves shifting the equilibrium between its
synthesis and destruction. The controlling steps occur at least partially in the
link between rhodopsin bleaching and phosphodiesterase activation, for Abramson et al. 2 have found that phosphodiesterase activation in minimally disrupted
outer segments occurs over 3-4 log units of light intensity. Phosphodiesterase
activity, like cyclic GMP levels, reaches and maintains a constant value at
intermediate levels of continuous illumination, and is related to the rate of
rhodopsin bleaching (to light intensity) rather than the absolute number of
rhodopsin molecules bleached. Part of the light-induced lowering of cyclic GMP
levels could also derive from inhibition of the guanylate cyclase activity. Krishna
et al. (1976) have reported a two-fold inhibition of guanylate cyclase caused by
illumination. Goridis and Weller (1976), however, failed to confirm this observation, and Abramson et al.2 find no effect of illumination.
The return of cyclic GMP levels to their dark values after brief illumination
probably is caused by deactivation of the phosphodiesterase. Abramson et al. 2
have observed an inactivation of phosphodiesterase after brief illumination, and
Wheeler and Bitensky (1977) have isolated a GTPase activity which they suggest
is involved in both activation and inactivation of phosphodiesterase. Also, it has
been shown that after large rhodopsin bleaches the regeneration of rhodopsin
by addition of l l-cis-retinal causes partial inactivation of phosphodiesterase
(Keirns et al., 1975; S. Green and D. Bownds3). It is difficult to determine
whether this mechanism of inactivation is used at the low light intensities
employed in these experiments (which bleached < 1% of the rhodopsin present),
for the frog rod outer segments have an endogenous store of l l-cis-retinal
sufficient to regenerate rhodopsin after a 1-3% bleach (Azuma et al., 1977). An
Published May 1, 1979
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THE JOURNAL
OF GENERAL
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4 Woodruff,M. L., and D. Bownds. Unpublishedobservation.
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attempt to enhance recovery of cyclic GMP levels after the cessation of
illumination by adding exogenous ll-c/s-retinal was not successful (Woodruff
and Bownds4).
The kinetics and stoichiometry of the light-induced cyclic GMP decrease are
consistent with a role for cyclic GMP in visual transduction. One possibility is
that cyclic GMP functions as an internal transmitter mediating between photon
absorption and the permeability decrease of the plasma membrane, a role
previously suggested for calcium ions. If the presence of cyclic GMP were
required to maintain high permeability (to "hold open channels"), its disappearance would lead to a suppression of permeability and photoreceptor hyperpolarization. Another possibility is that cyclic GMP plays an important role in
visual adaptation, with changes in cyclic GMP modifying the effect of illumination on the permeability mechanism.
Following the suggestion of Hagins and Yoshikami that calcium might be an
internal transmitter in outer segments, considerable interest has focused on
measuring the physiological effects of low and high calcium conditions (see, for
example, Hagins and Yoshikami, 1974; Brown et al., 1977; Lipton et al., 1977 a;
Wormington and Cone, 1978). There is general agreement that permeability of
rod receptor cells is suppressed if calcium concentration is raised to 3-10 raM,
and thus raising external calcium appears to mimic the effect of illumination.
Conversely, dark permeability increases when calcium concentration is lowered
by the addition of the chelating agent EGTA. Experiments using calcium
ionophores or intracellular injections of" calcium confirm that these permeability
effects result from changes in intracellular calcium levels. Although there is
agreement that both intracellular calcium and light can influence outer segment
permeability, Bastian ~1978) has provided evidence that calcium and light have
very different effects on the sensitivity and waveform of the rod responses, and
similar observations have been made for cone responses (Bertrand et al., 1978).
It is possible that the effect of exogenous calcium on permeability may derive
from the calcium-induced changes in cyclic GMP concentration. Cyclic GMP
levels, like permeability, are suppressed by calcium addition. The locus of this
calcium effect may be the guanylate cyclase, for Troyer et al. (1978) and
Krishnan et al. (1978) have demonstrated that calcium ions can inhibit this
enzyme. Miki et al. (1973) have failed to note an effect of calcium on phosphodiesterase activity. Further work will be required to determine whether calcium
effects other photoreceptor chemistry, and how calcium and cyclic GMP might
interact in controlling rod outer segment permeability. Interaction between
calcium and cyclic nucleotides as a physiological control mechanism has been
suggested for other cell systems (for review, see Rasmussen and Goodman,
1977).
In other neural systems, it has been speculated that cyclic nucleotides control
ion permeability by regulating the covalent modification of proteins, most
notably through phosphorylation/dephosphorylation sequences (Greengard,
1978). If this were the case in rod outer segments, one might expect to find
protein phosphorylation which is maximal in the dark, when cyclic GMP levels
Published May 1, 1979
WOODRUFFANDBOWNDS Light-Induced Decrease in Cyclic GMP: Characteristics
651
are high, and d e p h o s p h o r y l a t i o n in the light as cyclic GMP levels decrease. This
laboratory has identified two low molecular weight protein c o m p o n e n t s o f f r o g
rod o u t e r segments which u n d e r g o a d e p h o s p h o r y l a t i o n / p h o s p h o r y l a t i o n seq u e n c e after brief illumination (Polans et al., 1978, Polans et al?). Agents which
e n h a n c e cyclic GMP levels a n d o u t e r segment permeability increase the phosp h o r y l a t i o n level o f the proteins. Addition o f calcium ions, which suppresses
permeability and cyclic G M P levels in isolated o u t e r segment, causes rapid
d e p h o s p h o r y l a t i o n o f the proteins. Establishing f u r t h e r correlations between
permeability changes in o u t e r segments and the p h o s p h o r y l a t i o n - d e p h o s p h o rylation o f these proteins, as well as d e t e r m i n i n g the locus o f these proteins, are
i m p o r t a n t steps in elucidating the possible function o f cyclic GMP in visual
transduction.
Receivedfor publication 31 August 1978.
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We would like to thank Michael Biernbaum, Alexandra Shedlovsky, Bruce Abramson, Arthur
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